The present invention fits into the technical field of products used for the treatment and prevention of urinary tract infections.
More specifically, the present invention relates to fimbriae, isolated in a highly purified state from E. coli, that have been shown to be effective for the aforementioned purposes in animals. Said fimbriae are thus potentially useful for the prevention and treatment of the cited infections in humans.
The E. coli strains used are Escherichia coli CECT 4484 and Escherichia coli CECT 4485 deposited by the applicant in the Spanish Culture Type Collection (Coleccixc3x3n Espaxc3x1ola de Cultivos Tipoxe2x80x94CECT) of the University of Valencia Campus de Burjaset, L41600 BURJASET, Valencia, Spain on Jan. 19, 1994, in accordance with the xe2x80x9cBudapest Treaty on International Recognition of the Deposit of Microorganisms for the purposes of the Process in Patent Materialxe2x80x9d. Both are feasible strains for xe2x80x9cReseeding in suitable culture mediaxe2x80x9d.
Escherichia coli is the most frequent etiological agent in infections of the urinary tract. Symptoms of such infection include acute and chronic cystitis, as well as pyelonephritis and asymptomatic bacteriuria ((1) xe2x80x9cProblemxc3xa1tica de las infecciones urinarias en Espaxc3x1axe2x80x9d (Problem of urinary infections in Spain) Liade, 1989; (2) Woodrow G C, Levine M M New generation vaccines, 1990).
Infections of the urinary tract are caused by Escherichia coli strains which contain adhesin structures that are capable of binding to the uroepithelial cells ((3) Johnson J W, Virulence factors in E. coli UTI. Clin. Microbiol. Reviews 1991; 4; 80-128). Said structures are one of the main causes of pathogenicity of these organisms in the urinary tract (3). These structures are comprised of protein subunits with a tubular shape that contain a terminal and/or side region where they combine with the receptor ((3) and (4) Brinton C C. The structure, function, synthesis and genetic control of bacterial pili and molecular model for DNA and RNA transport in gram negative bacteria. Trans NY Acad. Sci. 1965; 27; 1003-1054). Said structures are named fimbriae and several families which are associated with factors of virulence, such as mannose resistant fimbriae (type P:P, F or NAP, type X or Dr related; 5, M, 6) and mannose sensitive fimbriae (type 1), have been identified (3). Type X, AFA-I and AFA-III mannose resistant adhesins are not associated with fibrial structures. Type 1 and Type P fimbriae have been tested for use as agents to prevent and/or protect against infection of the urinary tract by uropathogenic E. coli ((5) Hagberg L, Leffler H, Svanborg-Eden C. Non-antibiotic prevention of UTI: Infection 1985; 13 (Suppl. 2): 196-200; (6) O""Hanley P. Lark D, Falkow S, Schoolnik G. Molecular basis of E. coli colonization of the upper urinary tract in Balb/c mice.xe2x80x94J. Clin. Invest. 1985; 75: 347-360; (7) Hutlgren S J, Porter T N, Schaeffer A J, Duncan J L. Role of type I pili and effects of phase variation on lower UTI produced by E. colo. Infect. Immun. 1985; 50: 370-377; (8) Silverblatt F S, Weinstein R. Rene P. Protection against experimental pyelonephritis by antibodies to pili. Science J. Infect Dis 1982; suppl. 33: 79-82).
The object of the present study was to purify fimbriae-adhesins of uropathogenic E. coli from urine samples of women with recurrent cystitis and to use the fimbriae as a product for preventive treatment of infections of the urinary tract.
Studies have already been conducted on the isolation of E. coli fimbriae and on the effectiveness of the use of fimbriae in vaccines for the prevention of urinary tract infections. Thus, aside from the publication of Pecha et al. ((9) Pecha B, Low D. O""Hanley P. Gal-Gal pili vaccines prevent pyelonephritis by piliated E. coli in murine model. J. Clin. Invest. 1989; 83:2102-2108), the following documents may be cited:
Patent DE3832785 regarding the isolation of p-adhesin from E. coli cells and use of the same for immunization, production of antibodies and diagnosis.
Patent EP-342173 regarding a new fibronectin bonding protein expressed as fimbria on E. coli and hybrid DNA encoding sequences, to prevent E. coli infections by means of vaccination or topical application.
Patent EP-314224 regarding a vaccine for the protection of fowl against septicemia by E. coli that contains immunogenic sections or type F11 fimbriae or antibodies against them.
Patent EP-249739 regarding a vaccine for immunization against infections of the urinary tract that comprise fimbriae with fimbriae of the same fimbric family as the infecting organisms.
Patent EP-179887 regarding an antigen that comprises a polypeptide adhesin determinant, useful in immunization against bacterial infections and in the production of antibodies for diagnosis of infections.
Patent EP-161095 regarding vaccines against urinary tract infections containing fragments of proteins of gal-gal fimbriae of E. coli. 
U.S. Pat. No. 0,546,584 regarding the preparation of vaccines from Escherichia coli with pharmacological uses to prevent or cure gastroenteritis.
In contrast to the disclosures of the above-cited documents, the present invention relates to the preparation and purification of Type 1 and Type P fimbriae-adhesins from E. coli strains isolated from the urine of women with recurrent cystitis. By means of the present invention it is possible to obtain intact fimbriae-adhesins, free from other proteins or structures that could affect the metabolism or localization of the product after administration to a subject, such as flagella, hemolysin and LPS. This ensures the elimination of toxic substances that may be associated with the final product.
The absence of cross reactivity between type 1 fimbriae-adhesins and type P fimbriae-adhesins allows the separate production of immunogens to each of the types of fimbriae. The two types of fimbria-adhesins may also be used to produce immunogens at equal concentrations.
Verification of the adherence capacity of the E. coli strains associated with the fimbriae allows one to work with a material that induces an immune-response against the adhesin fraction that causes the fixation of E. coli to uroepithelial cells.
In one aspect, the present invention relates to composition comprising a member selected from the group consisting of an intact Type P fimbriae isolated from E. coli CECT 4484, an intact Type I fimbriae isolated from E. coli CECT 4485, and mixtures thereof, where the fimbriae have a molecular weight of between 2xc3x97105 and 2xc3x97107 daltons and are comprised of 90-95% by weight of protein and 1-3% by weight of sugar, each of the fimbriae comprising protein fractions of 5 different molecular weights, wherein the five protein fractions of type 1 fimbriae have molecular weights of between 14 and 20 kDa, with a fraction of the type 1 fimbriae with a molecular weight of 17 to 18 kDa comprising about 55% of the protein of the type 1 fimbriae, wherein the five protein fractions of type P fimbriae have molecular weights between 14 and 20 kDa and comprise a fraction having a molecular weight of 19 to 20 kDa wherein the fraction comprises about 35% of the protein of the type P fimbriae, wherein the 17 to 18 kDa fraction of the type 1 fimbriae and the 19 to 20 kDa fraction of the type P fimbriae are linked to carbohydrates, wherein the carbohydrates contain mannose-mannose units xcex1(1-3), xcex1(1-6), or xcex1(1-2), xcex1 sialic acid, xcex1(2-6), or xcex1(2-3) galactose, galactose xcex2(1-3) n-acetyl galactosamine, and galactose xcex2(1-4) n-acetyl glucosamine.
In another aspect, the invention is directed to methods for the purification of fimbriae adhesins from strains CECT 4484 and CECT 4485 of E. coli, the process comprising:
a) seeding bacteria of strains CECT 4484 and CECT 4485 of E. coli in a liquid culture medium at about 37xc2x0 C. until cultures are obtained at the beginning of the stationary phase (108-109 ucf/ml);
b) centrifuging the cultures and washing the obtained sediment;
c) resuspending the sediments washed in a physiological saline solution with a neutral pH and cold homogenizing same in a shear homogenizer for 2 to 10 minutes, at a speed of 10,000 to 25,000 rpm;
d) centrifuging the homogenates thus obtained at some 25,000-45,000xc3x97g for 20-45 minutes, discarding the sediment containing the bacteria and retrieving the supernatant liquid;
e) subjecting the supernatant liquid containing the fimbriae to several cold saline precipitations for a time period between 2 and 15 hours, retrieving the precipitate by centrifugation;
f) reconstituting the precipitate thus obtained in an aqueous physiological solution of a neutral pH and dialyzing the obtained solution;
g) treating the dialyzed solution containing the fimbriae with sodium deoxycholate 5% at about 80xc2x0 C.;
h) subjecting the product resulting from the previous step to chromatography in a gel filtration column containing Sephacryl S-200 and subjecting the chromatographed product located in the exclusion volume to another chromatography in a column containing Sepharose 4B, and collecting the inclusion volume to obtain the fimbriae.
In yet another aspect, the invention is directed to methods for the prevention and treatment of urinary tract infections caused by fimbriated E. coli.